Membrane fusion is required for important biological processes such as neurotransmitter release at the synapse and secretion of insulin from the pancreatic p cells. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are the core constituents of the intracellular fusion machinery which is highly conserved from yeast to human. Formation of the SNARE complex between vesicle-associated (v-) SNARE and target membrane (t-) SNARE bridges two membranes, facilitating fusion. The long-term goal of this project is to elucidate the molecular mechanism by which SNAREs mediate membrane fusion. The present project uses site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR), an established technique for the investigation of structures and topologies of membrane proteins. On the basis of various EPR results, a structural model of the protein in the native-like phospholipid bilayer is generated at the backbone resolution. In this project, structures and membrane topologies of individual full-length SNARE proteins and the final complex are determined using SDSL EPR. In particular, emphasis is placed on the transmembrane domains that are directly involved in driving lipid mixing and fusion pore formation. SNARE-mediated membrane fusion may involve multiple intermediates. The newly developed single membrane fusion assay based on total internal reflection (TIRF) microscopy makes it possible to dissect and characterize individual intermediate steps along the fusion pathway in unprecedented detail. In this project, the full dynamics of lipid mixing during fusion will be monitored in real time using this new generation fusion assay. Our studies will be focused on the SNARE system involved in trafficking from Golgi to the plasma membrane in yeast. Amino acid sequences of SNAREs implicated in various endocytic and secretory pathways are very similar to one another. Therefore, what is learned from yeast SNAREs will have general implications. Dysfunction of SNARE assembly and membrane fusion results in severe mental illness and diabetes. Thus, this study will help us to understand the molecular mechanism of SNARE-related illnesses.